| dc.description.abstracteng | Synaptotagmin-1 is a calcium dependent trigger of the SNARE-fusion machinery
that ultimately upon stimulation executes signal transmission between
neurons. However, the actual mechanistic function and the role of different
calcium binding domains of synaptotagmin-1 still remain unclear. The original
motivation for this study was therefore based on interesting findings and suggestions
of three previous publications addressing the role of synaptotagmin-1
in exocytosis [121, 119, 83]. In one of these studies, it has been proposed that
the triggering function of synaptotagmin-1 is based on a membrane distance
regulation mechanism [119]. In addition, it has been shown that cis-membrane
binding of synaptotagmin-1 is favoured over trans-binding [121], but that fusion
nonetheless can be enhanced by ATP [83]. It remains unclear whether ATP prevents
cis-binding directly or acts at a later stage by just pushing the equilibrium
further towards the fused state. Furthermore, the role of synaptotagmin-1 in
exocytotic priming and triggering is still elusive [49]. Here, two models have been
proposed with each of them accounting for conflicting findings that have been
made in the past [49].The authors of the above mentioned articles used different
fluorescence techniques like fluorescence anisotropy [83], Förster-resonanceenergy-
transfer [119] and Fluorescence-cross-correlation-spectroscopy (FCCS)
[121]. The present study aims to find further evidence for or against a distance
regulation function of synaptotagmin-1 with the help of additional FCCS studies
combined with fluorescence lifetime analysis. In fact, the results presented
in this thesis demonstrate that synaptotagmin-1 can act as a distance regulator
when bound to PiP2 by synchronous alterations in tethering and membrane
distance shortening. Here, synchronous tethering to an opposing lipid bilayer
is furthermore achieved by the C2B domain of synaptotagmin-1. Calcium ions,
which are necessary for tethering, trigger this PiP2 dependent distance regulation
function. Despite the cooperative behaviour of synaptotagmin-1 towards
binding Ca2+ in the presence of PiP2 [118], a reduction of the intermembrane
distance occurs proportional to tethering. However, high PiP2 concentrations
increase the base level of tethering. Moreover, the distance regulation function
of synaptotagmin-1 is circumvented by the presence of syntaxin 1A.
In addition, a new approach to study tethering and fusion processes by
optical trapping is introduced. This method reveals the first way of studying
exocytotic processes at plasma membrane (PLM) analogues without introducingany artifacts like an enhanced surface tension. It is demonstrated that optical
trapping of large unilamellar vesicles (d= 110 nm) is possible when filled with
2 M sucrose. Here, the combination of a CW and a TP laser beam, both
operating at 800 nm, permits for super-position of the trapping center with the
TPE-volume. Hence, trapping and FCS can be combined, permitting to study
processes in real-time. A conceivable application arising from the combination
of both laser beams is to study retention times of liposomal tethering which may
be analyzed in terms of adsorption isotherms. Furthermore, fusion to a single
LUV can be monitored on a min timescale. | de |